Progress in C-terminal sequencing of peptides and proteins has been slow and painstaking over the last several decades in comparison to N-terminal peptide sequencing, e.g. Inglis, Analytical Biochemistry, 195:183-196 (1991). An important approach to C-terminal sequencing involves the formation of a thiohydantoin moiety at the C-terminus of the peptide or protein sample, which is then cleaved and detected in much the same manner that a phenylthiohydantoin moiety is formed, cleaved, and detected in the Edman degradation approach to analyzing N-terminal amino acid residues.
Much of the current research into C-terminal sequencing methodologies has focused on the development of better reagents for thiohydantoin formation, e.g. Bailey et al, Biochemistry, 29:3145-3156 (1990); Boyd et al, U.S. Pat. No. 5,051,368; Bailey et al, U.S. Pat. No. 5,180,807; Boyd et al, U.S. Pat. No. 5,304,497; and the like. A variety of chemical methods have been proposed for converting the C-terminal amino acid of a peptide to its corresponding thiohydantoin (TH), e.g. Inglis (cited above). In particular, Stark and others have employed acetic anhydride under acidic conditions to activate the C-terminal residue, which is concurrently treated with thiocyanate {N.dbd.C.dbd.S }.sup.- to produce a thiohydantoin derivative, e.g. Stark, Biochemistry, 7:1796-1807 (1968). The chemical mechanism underlying such activation and thiohydantoin formation is not well understood, but it is believed to involve the formation of mixed anhydrides and oxazolones. Unfortunately, the oxazolones are thought to participate in several undesirable side reactions which tends to reduce the yield of thiohydantoin.
It would be advantageous if there were available an alternative chemistry for forming thiohydantoins in C-terminal peptide sequencing that resulted in higher thiohydantoin yields and that could thereby support a greater number of sequencing cycles on reasonably sized samples.